Endocytosis Is Essential for Pathogenic Development in the Corn Smut Fungus Ustilago maydis

Abstract

It is well established that polarized exocytosis is essential for fungal virulence. By contrast, the contribution of endocytosis is unknown. We made use of a temperature-sensitive mutant in the endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptor Yup1 and demonstrate that endocytosis in Ustilago maydis is essential for the initial steps of pathogenic development, including pheromone perception and cell-cell fusion. Furthermore, spore formation and germination were drastically reduced, whereas colonization of the plant was only slightly inhibited. The function of endocytosis in the recognition of mating pheromone through the G protein-coupled pheromone receptor Pra1 was analyzed in greater detail. Biologically active Pra1-green fluorescent protein localizes to the plasma membrane and is constitutively endocytosed. Yup1 ts mutants that are blocked in the fusion of endocytic transport vesicles with early endosomes are impaired in pheromone perception and conjugation hyphae formation. This is attributable to an accumulation of Pra1-carrying endocytic vesicles in the cytoplasm and the depletion of the receptor from the membrane. Consistently, strong Pra1 expression rescues the signaling defects in endocytosis mutants, but subsequent cell fusion is still impaired. Thus, we conclude that endocytosis is essential for recognition of the partner at the beginning of the pathogenic program but has additional roles in mating as well as spore formation and germination.

Polar Growth in the Infectious Hyphae of the PhytopathogenUstilago maydisDepends on a Virulence-Specific Cyclin

“…In agreement with some role of Pcl12 in the formation of the conjugation tubes, we found that pcl12 was induced after pheromone treatment ( Figure 7C). These data suggest that Pcl12 plays a role in the polar growth of the conjugation tube, although we cannot exclude some indirect effect such as the possibility that Pcl12 participate in pheromone sensing as it has been recently proposed for Myo5 and Yup1 (Weber et al, 2003;Fuchs et al, 2006). To address this issue, we took advantage of the fuz7DD allele, which encodes an activated version of the mitogenactivated protein kinase (MAPK) kinase involved in pheromone response (Banuett and Herskowitz, 1994;Mü ller et al, 2003), since cells expressing this allele under the arabinose-induced crg1 (D) Pcl12-induced filamentation is dependent on cdk5.…”

Transient binding of dynein controls bidirectional long-range motility of early endosomes

“…In this fungus, the only membranous cargo known to travel in a bidirectional fashion along MTs is EEs (23). Similar to that in humans (24) and amoebas (10), kinesin-3 motors carry the EEs to MT plus ends at the hyphal tip (anterograde motility) (25,26), where they support polarized growth and mating by local endocytic recycling (23,27). The importance of the retrograde dynein-driven EE motility is unknown, but it might mediate communication with the nucleus (28).…”

“…If the apical dynein accumulation serves as a loading zone, all EEs have to reach the hyphal tip before changing direction. We tested this by visualizing anterograde motility of photoactivatable green fluorescent protein (paGFP) fused to the small endosomal GTPase Rab5a, which has been shown to localize to EEs in U. maydis (27). The use of this GFP variant enabled us to visualize subsets of EEs and reduced interferences.…”

Tandem KH domains of Khd4 recognize AUACCC and are essential for regulation of morphology as well as pathogenicity inUstilago maydis

“…Promising candidate mRNAs for Khd4 that contain AUACCC, but were not detected as differentially expressed in our microarray analysis, are cdc42, yup1, and spa2, encoding a small G protein, a putative t-SNARE, and a polarisome component, respectively (Wedlich-Söldner et al 2000;Mahlert et al 2006;Carbo and Perez-Martin 2008). Deletion of cdc42 leads to defects in cytokinesis and virulence (Mahlert et al 2006), loss-of-function mutants in yup1 are affected in cell morphology and virulence (Wedlich-Söldner et al 2000;Fuchs et al 2006), and spa2D strains exhibit increased cell diameters and form shorter filaments, albeit deletion strains are fully pathogenic (Carbo and Perez-Martin 2008). Another candidate for Khd4 regulation is AUACCC-containing mRNA clp1 encoding a key factor of clamp formation, which is essential for pathogenicity.…”

“…strain SG200R (SG200 variant expressing cytoplasmic mRfp, monomeric version of the red fluorescent protein) (Bölker et al 1995;Campbell et al 2002;Fuchs et al 2006). This strain enables plant infection experiments without prior mating.…”